Method for detecting carcinoma and agent for suppressing carcinoma

ABSTRACT

An object of the present invention is to identify genes exhibiting characteristic behavior in the cases of carcinoma such as oral squamous-cell carcinoma using a change of expression level of micro RNA in oral squamous-cell carcinoma as an indicator, so as to provide a method for detecting carcinoma and an agent for suppressing carcinoma. The present invention A method for detecting oral squamous-cell carcinoma, which comprises detecting malignant transformation of specimens by employing a change of expression level of micro RNA as an indicator, and an agent for suppressing oral squamous-cell carcinoma using micro RNA.

TECHNICAL FIELD

The present invention relates to a method of detecting carcinomas, suchas oral squamous-cell carcinoma by utilizing changes in the expressionlevels of microRNA genes that are present in the human chromosome, andmore particularly to an agent for suppressing growth of carcinoma, suchas oral squamous-cell carcinoma.

BACKGROUND ART

Oral squamous-cell carcinoma (OSCC) is classified as a head and neckcarcinoma and is a tumor that is mainly generated from oral mucousmembrane epithelia and the like. Among head and neck carcinomas, OSCCincidence is as high as about 35% and it is assumed to develop in about270,000 people worldwide every year (Parkin, D. M., et al., CA Cancer J.Clin. 55, 74-108, 2002). In Japan, 5,500 or more people had died thereofin 2003. The most common site of the origin of oral squamous-cellcarcinoma is tongue and the second most common site thereof is gingiva(gum). Oral squamous-cell carcinoma is known to be developed at othermucous membranes of the oral cavity such as buccal mucosa, palate, andmouth floor. Furthermore, oral squamous-cell carcinoma is also known tobe developed at jawbone or salivary gland.

In recent years, although methods for diagnosis and treatment for oralsquamous-cell carcinoma have been advanced, the prognosis thereof hasremained unimproved. Accordingly, it has been necessary to discover acausative gene for oral squamous-cell carcinoma and changes that haveoccurred in such gene to elucidate the functions for establishment ofnew therapeutic strategy for development of more effective therapeuticmethods and chemical prevention.

DISCLOSURE OF THE INVENTION

Successful elucidation of the mechanism of malignant transformation oforal-cavity-derived cells and mainly oral mucous membraneepithelium-derived cells at the gene level will enable detection ofmalignant transformation of oral mucous membrane epithelium cells at thegene level, diagnosis of the malignancy of oral squamous-cell carcinoma,and suppression of the advancement thereof. Furthermore, it will alsoenable establishment of methods for selecting and developing drugs andmethods for therapy, based on such mechanisms. Specifically, identifyinggenes exhibiting characteristic behavior observed in oral squamous-cellcarcinoma cases and then carrying out technical examination mainlytargeting such genes can achieve this object. Hence, an object to beachieved by the present invention is to identify genes exhibitingcharacteristic behavior in the cases of carcinoma such as oralsquamous-cell carcinoma, so as to provide a method for detectingcarcinoma and an agent for suppressing carcinoma.

The real-time reverse transcription-polymerase chain reaction (Real-timeRT-PCR) is the best method for conveniently and rapidly analyzing theamount of transcripts resulting from gene expression. In order toanalyze changes in expression of genes associated with carcinoma, TaqManMicroRNA Assays was used, and the carcinoma-associated microRNA genes,which would accelerate malignant transformation of oral mucous membraneepithelium cells, shown in Tables 1 and 2 were successfully identified.The expression levels of miR-34b, miR-132, miR-137, miR-193a, andmiR-203 located on or around the CpG islands are down-regulated also bymethylation of cytosine that is present on CpG islands. Specifically,down-regulated expression levels of such genes result in acceleration oforal squamous-cell carcinoma growth. Further, the present inventorsdiscovered that the up-regulated expression levels of such genes in oralsquamous-cell carcinoma would significantly down-regulate carcinomagrowth. This has led to the completion of the present invention.

The present invention provides a method for detecting carcinoma, whichcomprises detecting malignant transformation of specimens by employingan up-regulated expression level of at least one gene selected from thegroup consisting of miR-374, miR-340, miR-224, miR-10a, miR-140,miR-213, miR-146a, miR-126, miR-31, miR-9, and miR-9*, and/or adown-regulated expression level of at least one gene selected from thegroup consisting of miR-27a, miR-34b, miR-34c, miR-203, miR-302c*,miR-23a, miR-27b, miR-34a, miR-215, miR-299, miR-330, miR-337, miR-107,miR-133b, miR-138, miR-139, miR-223, miR-204, miR-370, let-7d, miR-95,miR-302a, miR-367, let-7g, miR-23b, miR-128a, miR-148a, miR-155,miR-200c, miR-302b, miR-368, miR-122a, miR-371, let-7a, miR-26b,miR-30e-5p, miR-96, miR-125a, miR-132, miR-200b, miR-199b, miR-296,miR-373*, miR-137, miR-197, miR-193a, let-7e, miR-30d, miR-331, miR-342,miR-338, miR-199a, miR-372, and miR-184 as indicators.

Preferably, changes in gene expression are detected by using the DNAarray method, Northern blotting, RT-PCR, real-time RT-PCR, RT-LAMP, orin situ hybridization.

Preferably, the down-regulated gene expression level results frommethylation of CpG islands or in the vicinity thereof.

Preferably, the methylation is detected by using the COBRA method,bisulfite sequencing, or Southern blotting.

Preferably, the down-regulated expression level of at least one geneselected from among miR-34b, miR-132, miR-137, miR-193a, and miR-203 isemployed as an indicator to detect malignant transformation ofspecimens.

Preferably, the specimen is an oral-cavity-derived cell.

Preferably, the carcinoma is oral squamous-cell carcinoma.

According to another aspect, the present invention provides an agent forsuppressing carcinoma, which comprises at least one gene selected fromthe group consisting of miR-27a, miR-34b, miR-34c, miR-203, miR-302c*,miR-23a, miR-27b, miR-34a, miR-215, miR-299, miR-330, miR-337, miR-107,miR-133b, miR-138, miR-139, miR-223, miR-204, miR-370, let-7d, miR-95,miR-302a, miR-367, let-7g, miR-23b, miR-128a, miR-148a, miR-155,miR-200c, miR-302b, miR-368, miR-122a, miR-371, let-7a, miR-26b,miR-30e-5p, miR-96, miR-125a, miR-132, miR-200b, miR-199b, miR-296,miR-373*, miR-137, miR-197, miR-193a, let-7e, miR-30d, miR-331, miR-342,miR-338, miR-199a, miR-372, and miR-184.

Preferably, the agent for suppressing carcinoma according to the presentinvention comprises at least one of miR-34b, miR-132, miR-137, miR-193a,and miR-203 genes.

Preferably, the gene is bound to or incorporated in a polymer compound.

Preferably, the polymer compound is a liposome.

Preferably, the carcinoma is oral squamous-cell carcinoma.

The present invention enables accurate understanding of malignanttransformation in cell specimens derived from the oral mucosa. Also,introduction of synthetic double-stranded RNA containing the microRNAsequence into the oral squamous-cell carcinoma would be able to suppressthe growth of carcinoma, such as oral squamous-cell carcinoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the strategy of the study and the results of miRNAexpression analysis in the oral squamous-cell carcinoma cells. FIG. Ashows a strategy for isolating antioncogenic miRNA, the expression ofwhich is epigenetically suppressed in the oral squamous-cell carcinomacells. FIG. B shows expression profiles of 157 types of miRNA genes in18 types of oral squamous-cell carcinoma cells and in the RT7 cellsobtained with the use of the TaqMan Micro RNA Assays Human Panel EarlyAccess Kit; wherein SS represents TaqMan Micro RNA Assays, sales ofwhich have been suspended; and U represents miRNA, the expression ofwhich was not observed in the RT7 cells. The miRNA expression levelswere compared in terms of relative value based on the RNU6B expressionlevel as an internal control. Stars represent 21 types of miRNA geneslocated on or around the CpG islands. FIG. C shows expression levels ofcandidate miRNA genes located on or around the CpG islands in 18 typesof oral squamous-cell carcinoma cells. A bar graph representing eachcell line represents a comparison of expression levels in the RT7 cellsand in each cell line. Asterisks (*) represent a frequency of oralsquamous-cell carcinoma cell lines in which down-regulated expressionlevels (expression levels of less than 0.5-fold) of candidate miRNA,compared with the RT7 cells, had been observed. FIG. D shows recovery ofcandidate miRNA gene expression after treatment with 10 μM 5-aza-dCyd. Abar graph representing each cell line indicates a comparison ofexpression levels in untreated cells and in treated cells. Starsrepresent cell lines in which significantly down-regulated geneexpression was not observed via TaqMan real-time RT-PCR analysis (FIG.1C). Asterisks (*) represent a frequency of oral squamous-cell carcinomacell lines in which recovery of candidate miRNA expression (expressionlevels of more than 1.5-fold) was observed by comparing untreated cellswith cells that have been treated with 10 μM 5-aza-dCyd.

FIG. 2 shows the correlation analysis of the extent of methylation andexpression of five types of candidate miRNA in the oral squamous-cellcarcinoma cells. FIG. A is a map showing the physical relationship ofmiRNA, CpG islands, the CpG sites, and PCR products used for the COBRAmethod and the bisulfite sequencing in the genome. A dark gray boxrepresents CpG islands; a light gray box represents an miRNA gene, avertical line represents a CpG site, a horizontal arrow represents a PCRproduct, and a vertical arrow represents a restriction enzyme site. Thesize of the PCR product in the miR-34b region 1 was 428 bp (cleaved withBstUI), that in the miR-34b region 2 was 549 bp (cleaved with BstUI);that in the miR-132 region 1 was 453 bp (cleaved with BstUI), that inthe miR-132 region 2 was 599 bp (cleaved with BstUI), that in themiR-132 region 3 was 442 bp (cleaved with BstUI), that in the miR-132region 4 was 408 bp (cleaved with BstUI); that in the miR-137 region 1was 444 bp (cleaved with TaqI); that in the miR-193a region 1 was 524 bp(cleaved with BstUI), that in the miR-193a region 2 was 522 bp (cleavedwith TaqI), that in the miR-193a region 3 was 458 bp (cleaved withTaqI); that in the miR-203 region 1 was 405 bp (cleaved with BstUI),that in the miR-203 region 2 was 655 bp (cleaved with BstUI), and thatin the miR-203 region 3 was 287 bp (cleaved with BstUI). FIG. B showsthe results of COBRA of the oral squamous-cell carcinoma cell lines andthe RT7 cells. Expression patterns of candidate miRNA in 18 types oforal squamous-cell carcinoma cell lines are shown above the results ofCOBRA. An arrow represents a nonmethylated allele, an arrowheadrepresents a methylated allele, a star represents a sample in which arestriction enzyme fragment derived from a methylated allele has beendetected, and asterisks (*) represent frequency of oral squamous-cellcarcinoma cell lines in which the extent of DNA methylation of candidatemiRNA was in accord with down-regulation of expression levels. FIG. Cshows the results of bisulfite sequencing for the RT7 cells, thecandidate miRNA expression-positive cell lines (+), and the candidatemiRNA expression-negative cell lines (−). A map showing the physicalrelationship of miRNA, CpG islands, the CpG sites, and PCR products usedfor the COBRA method and the bisulfite sequencing in the genome is shownabove the results of bisulfite sequencing. A light gray box representsan miRNA gene, a vertical line represents a CpG site, a horizontal arrowrepresents a PCR product, and a vertical arrow represents a restrictionenzyme site. White and black squares each show nonmethylated andmethylated CpG sites, and a lane is derived from a single type of clone.

FIG. 3 shows the methylation analysis and the expression analysis ofcancerous and non-cancerous tissue for 11 cases of patients with oralsquamous-cell carcinoma. FIG. A shows the results of COBRA for thecandidate miRNA gene of cancerous sites (T) of surgically removed oralsquamous-cell carcinoma from 11 patients and for correspondingnoncancerous tissue (N). Whether or not the cells have been treated witha restriction enzyme is indicated by the symbol “+” or “−” above theresults of COBRA. A star represents a case in which cancer-specifichypermethylation has been detected. Asterisks (*) represent frequency ofcancer-specific hypermethylation of candidate miRNA determined viaCOBRA. FIG. B shows the results of quantitative real-time RT-PCRanalysis of candidate miRNA expression in cancerous sites and innoncancerous tissue obtained from 11 patients with oral squamous-cellcarcinoma. A star represents a case in which cancer-specifichypermethylation has been detected. Asterisks (*) represent frequency ofcases in which cancer-specific hypermethylation has been detected viaCOBRA and candidate miRNA expression is down-regulated in canceroustissue (expression levels of less than 0.5-fold), compared withnoncancerous tissue. FIG. C shows representative examples of the resultsof bisulfite sequencing. A horizontal arrow represents a PCR product, avertical arrow represents a restriction enzyme site, white and blacksquares each show nonmethylated and methylated CpG sites, and a lane isderived from a single type of clone.

FIG. 4 shows the effects of miR-137 and miR-193 a for suppressingcarcinoma in the oral squamous-cell carcinoma cell lines. FIG. A showsthe growth curve and the phase contrast microscopic photograph of oralsquamous-cell carcinoma cell lines after the transfection of 10 nM ofPre-miR™ miRNA Precursor Molecule or nonspecific miRNA as a controlusing Lipofectamine RNAiMAX. The viable cell counts 24 to 72 hours afterthe transfection were evaluated by WST assay. The results of theexperiments are shown in terms of the average (bar, SE) of three assayoperations at each data point. The phase contrast microscopic photographshows the cells that have been cultured for 72 hours after thetransfection. FIG. B shows a TUNEL stain image of HSC-2 cells 24 hoursafter the transfection of miR-193a or nonspecific dsRNA as a controlunder a fluorescent microscope. FIG. C shows apoptosis induction inHSC-2 cells 24 hours after the transfection of miR-193a or nonspecificdsRNA as a control.

BEST MODES FOR CARRYING OUT THE PRESENT INVENTION

Hereafter, the present invention is described in greater detail.

(1) Method for Detecting Cancer

The method for detecting carcinoma according to the present inventioncomprises detecting malignant transformation of specimens using at leastone of changes in expression of genes shown in Table 1 and Table 2 asindicators. The term “changes in expression of genes” used herein refersto up-regulation or down-regulation of gene expression. Particularlypreferably, down-regulated expression levels of microRNA (miRNA) genes(i.e., miR-34b, miR-132, miR-137, miR-193a, or miR-203) in specimens canbe detected to identify malignant transformation of specimens.Particularly preferably, down-regulated expression levels of miR-34b,miR-132, miR-137, miR-193a, and miR-203 genes in oral-cavity-derivedcells can be detected to identify malignant transformation oforal-cavity-derived cells. Further, miR-34b, miR-132, miR-137, miR-193a,and miR-203 can be bound to or incorporated in polymer compounds, andthe resultants can then be introduced into oral squamous-cell carcinomacells to suppress growth of carcinoma.

As a result of the human genome project etc., transcripts of themiR-34b, miR-132, miR-137, miR-193a, and miR-203 are already known viaTaqMan Micro RNA assays, and such transcripts are microRNA genes thatare located in chromosomal regions 11q23.1, 17p13.3, 1p21.3, 17q11.2,and 14q32.33. Detailed functions of miR-34b, miR-132, miR-137, miR-193a,and miR-203 genes remain unknown. The fact that such miRNA is animportant cancer-associated gene involved in the onset of oralsquamous-cell carcinoma was unknown before the present invention.

As described above, the detection method according to a preferableembodiment of the present invention comprises detecting down-regulatedexpression levels of miR-34b, miR-132, miR-137, miR-193a, and miR-203genes in oral-mucosa-derived cells or oral squamous-cell carcinoma.

Target oral-mucosa-derived cells or oral squamous-cell carcinoma cellsin which down-regulated expression levels of miR-34b, miR-132, miR-137,miR-193a, and miR-203 genes are to be detected are preferably biopsytissue cells obtained from a specimen donor.

Such tissue cell specimen may be an oral-cavity-derived cell of ahealthy subject or a cancerous tissue of an oral squamous-cell carcinomapatient. In practice, examples of a major target tissue specimen thatcan be used herein include: a tissue obtained from a lesion in whichsuspected malignant transformation of the mucous membrane of oralcavity, tongue, gum, or the like is observed by a test or the like; andan oral squamous-cell carcinoma tissue that has been confirmed to bederived from oral squamous-cell carcinoma and thus must be subjected todetermination of malignancy or the stage progression of oralsquamous-cell carcinoma.

In a case in which down-regulated expression levels of miR-34b, miR-132,miR-137, miR-193a, and miR-203 genes are observed in “a tissue obtainedfrom a lesion in which suspected malignant transformation oforal-cavity-derived tissues or cells is observed in a test or the like”by the detection method of the present invention, it is understood thatsuch lesion tissue will reach (or has reached) the state of malignanttransformation so that the level of malignancy of the disease willincrease. Thus, there is a demonstrated urgent need for implementationof a full-scale therapy (e.g., elimination of a lesion via a surgery orthe like and full-scale chemotherapy). In addition, in a case in whichthe down-regulated expression levels of miR-34b, miR-132, miR-137,miR-193a, and miR-203 genes are observed in “an oral squamous-cellcarcinoma tissue that has been confirmed to be derived from oralsquamous-cell carcinoma and thus must be subjected to determination ofmalignancy or the stage progression of oral squamous-cell carcinoma,” itis also understood that the level of malignancy of the cancerous tissuewill increase. Thus, there is a demonstrated urgent need forimplementation of full-scale therapy (e.g., elimination of a lesion viaa surgery or the like and full-scale chemotherapy). An oralsquamous-cell carcinoma tissue collected as a specimen may be subjectedto necessary treatment such as preparation of DNA or RNA from thecollected tissue followed by the detection method of the presentinvention.

The sequence information regarding microRNA shown in Tables 1 and 2 usedin the present invention has been registered in the Wellcome TrustSanger Institute miRBase (http://microrna.sanger.ac.uk/). The accessionnumbers thereof are shown below.

miRNA miRBase Accessions miR-374 MIMAT0000727 miR-340 MIMAT0004692miR-224 MIMAT0000281 miR-10a MIMAT0000253 miR-140 MIMAT0000431 miR-213MIMAT0000256 miR-146a MIMAT0000449 miR-126 MIMAT0000445 miR-31MIMAT0000089 miR-9 MIMAT0000441 miR-9* MIMAT0000442 miR-27a MIMAT0000084miR-34b MIMAT0004676 miR-34c MIMAT0000686 miR-203 MIMAT0000264 miR-302c*MIMAT0000716 miR-23a MIMAT0000078 miR-27b MIMAT0000419 miR-34aMIMAT0000255 miR-215 MIMAT0000272 miR-299 MIMAT0002890 miR-330MIMAT0004693 miR-337 MIMAT0004695 miR-107 MIMAT0000104 miR-133bMIMAT0000770 miR-138 MIMAT0000430 miR-139 MIMAT0000250 miR-223MIMAT0000280 miR-204 MIMAT0000265 miR-370 MIMAT0000722 let-7dMIMAT0000065 miR-95 MIMAT0000094 miR-302a MIMAT0000684 miR-367MIMAT0000684 let-7g MIMAT0000414 miR-23b MIMAT0000078 miR-128aMIMAT0000424 miR-148a MIMAT0000243 miR-155 MIMAT0000646 miR-200cMIMAT0000617 miR-302b MIMAT0000715 miR-368 MIMAT0000720 miR-122aMIMAT0000421 miR-371 MIMAT0004687 let-7a MIMAT0000062 miR-26bMIMAT0000083 miR-30e-5p MIMAT0000692 miR-96 MIMAT0000095 miR-125aMIMAT0000443 miR-132 MIMAT0000426 miR-200b MIMAT0000318 miR-199bMIMAT0000263 miR-296 MIMAT0000690 miR-373* MIMAT0000725 miR-137MIMAT0000429 miR-197 MIMAT0000227 miR-193a MIMAT0004614 let-7eMIMAT0000066 miR-30d MIMAT0000245 miR-331 MIMAT0004700 miR-342MIMAT0004694 miR-338 MIMAT0004701 miR-199a MIMAT0000231 miR-372MIMAT0000724 miR-184 MIMAT0000454(2) Changes in miRNA Gene Expression in Oral Squamous-Cell Carcinoma

An example of a representative method that can directly detect changesin miRNA gene expression levels is real-time RT-PCR.

The method for detecting mature miRNA is preferably and practicallycarried out using TaqMan Micro RNA Assays (Applied Biosystems).Detection may be carried out via Northern blot analysis or the like,although TaqMan Micro RNA Assays is more convenient and sensitive.

(3) Detection of Down-Regulated Expression of miRNA Gene in OralSquamous-Cell Carcinoma

It has been reported that transcriptional inactivation occurs when aCpG-rich promoter region and an exon region are densely methylated (BirdA P., et al., Cell, 99, 451-454, 1999). In the cases of carcinoma cells,CpG islands are frequently and densely methylated compared with otherregions, and thus hypermethylation of a promoter region is deeplyinvolved in the inactivation of an antioncogene of a carcinoma (EhrlichM., et al, Oncogene, 21, 6694-6702, 2002). As described below, CpGislands actually exist in the vicinity of the miR-34b, miR-132, miR-137,miR-193a, and miR-203 genes. In addition, the extent of methylation ofthe CpG islands was strongly correlated with suppression of theexpression of the miR-34b, miR-132, miR-137, miR-193a, and miR-203 genesin some oral squamous-cell carcinoma cases. In addition, it was possibleto demethylate such CpG islands by culturing such oral squamous-cellcarcinoma cells in the presence of 5-aza-2′-deoxycytidine (5-aza-dCyd)serving as a demethylating reagent. As a result, it was possible torecover the expression levels of the miR-34b, miR-132, miR-137,miR-193a, and miR-203 genes. Based on the above results, it has beenrevealed that hypermethylation of CpG islands is a cause of frequentlyoccurring suppression of the expression of antioncogenic miRNA genes insquamous-cell carcinoma.

(4) Method for Suppressing Carcinoma, and Agent for SuppressingCarcinoma

The present invention further provides a method for suppressingcarcinoma which comprises introducing miRNA, which is a transcript ofthe miRNA gene (particularly preferably the miR-34b, miR-132, miR-137,miR-193a, or miR-203 gene), into cells, which are listed in Tables 1 and2 as genes, the expression levels of which are down-regulated in OSCCcells, and an agent for suppressing the carcinoma which comprises theabove gene.

The term “miRNA” refers to 10-bp or longer short double-stranded RNA,which is artificially and chemically synthesized, biologicallysynthesized, synthesized in vivo, or generated via degradation ofdouble-stranded RNA having about 40 or more nucleotides in vivo. Ingeneral, miRNA has a 5′-phosphoric acid or 3′-OH structure, and the 3′end is protruded by approximately 2 nucleotides.

The agent for suppressing carcinoma of the present invention can beprepared by incorporating the above gene as an active ingredienttogether with a base which is commonly used for an agent for genetherapy. When the above gene is incorporated into a virus vector, virusparticles containing recombinant vectors are prepared, and theresultants are incorporated with a base which is commonly used for anagent for gene therapy.

As a base used for incorporating the gene as an active ingredient, abase that is commonly used for an injection can be used. Examplesinclude distilled water, a salt solution of sodium chloride or a mixtureof sodium chloride and an inorganic salt, a solution of mannitol,lactose, dextran, or glucose, an amino acid solution of glycine orarginine, an organic acid solution, and a mixed solution of a saltsolution and a glucose solution. Alternatively, an adjuvant, such as aregulator of osmotic pressure, a pH adjuster, vegetable oil, or asurfactant, may be added to the base in accordance with a techniqueknown in the art to prepare an injection in the form of a solution,suspension, or dispersion. Such injection can be prepared in the form ofa preparation to-be-dissolved before use via operations, such aspulverization or lyophilization.

The agent for suppressing carcinoma of the present invention may beadministered systemically, for example, common intravenous orintraarterial administration. Alternatively, topical administration,such as topical injection or oral administration, may be employed.Further, the agent for suppressing carcinoma can be administered incombination with catheterization, gene introduction, surgery, or thelike. Specifically, the agent for suppressing carcinoma of the presentinvention may be administered orally, parenterally (e.g., intravenous,intramuscular, hypodermic, subcutaneous, mucosal, intrarectal,intravaginal, topical administration to a lesion, or percutaneousadministration), or via direct administration to the lesion. When theagent of the present invention is used in the form of a pharmaceuticalcomposition, a pharmaceutically acceptable additive can be mixedaccording to need. Specific examples of a pharmaceutically acceptableadditive include, but are not limited to, an antioxidant, apreservative, a colorant, a flavoring agent, a diluent, an emulsifier, asuspending agent, a solvent, a filler, an augmentor, a buffer, adelivery vehicle, a diluent, a carrier, an excipient, and/or apharmaceutical adjuvant.

The agent for suppressing carcinoma of the present invention may be usedin any form without particular limitation, and examples include, aliquid, an injection, and a controlled-release agent. A solvent used forpreparing the agent of the present invention into the above preparationmay be aqueous or nonaqueous.

Further, miRNA as an active ingredient of the agent for suppressingcarcinoma of the present invention can be administered via, for example,a method of using a liposome to introduce nucleic acid molecules (e.g.,the liposome method, the HVJ-liposome method, the cationic liposomemethod, the lipofection method, or the lipofectamine method),microinjection, or a method of using a gene gun to transfer nucleic acidmolecules together with carriers (i.e., metal particles) to the cells.When the agent is administered to a living body with the use of miRNA, avirus vector, such as a recombinant adenovirus or retrovirus vector, canbe used. The miRNA gene is incorporated into DNA or RNA viruses, such asdetoxicated retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, pox virus, poliovirus, Sindbis virus, Sendaivirus, or SV40, and the cell or tissue is infected with the recombinantvirus to introduce the gene of interest into cells or tissue.

A person skilled in the art can determine the dose of the agent forsuppressing carcinoma of the present invention in accordance with thepurpose of use, severity of the disease, the age, the body weight, sex,or a history of use of a patient, or a type of miRNA as an activeingredient. The dose of miRNA is not particularly limited. For example,it is approximately 0.1 ng to approximately 100 mg/kg/day, andpreferably approximately 1 ng to approximately 10 mg/kg/day. In general,the effects of miRNA appear 1 to 3 days after the administration. Thus,it is preferable that the agent be administered at a frequency of dailyto once every three days. When an expression vector is used,administration can be carried out approximately once in a week.

The present invention is hereafter described in greater detail withreference to the following examples, although the present invention isnot limited thereto.

EXAMPLES Example 1 Changes in miRNA Gene Expression in OralSquamous-Cell Carcinoma

To detect changes in miRNA gene expression in oral squamous-cellcarcinoma, 18 types of oral squamous-cell carcinoma cell lines (Ca9-22,HO-1-N-1, HSC-2, HSC-3, HSC-4, KOSC-2 c13-43, HOC-313, HOC-815, HSC-5,HSC-6, HSC-7, KON, NA, OM1, OM2, SKN3, TSU, and ZA) were used. As acontrol, a normal oral mucosal epithelium-derived immortalized cellline, RT7, was used. The oral squamous-cell carcinoma cell line wascultured in DMEM medium containing streptomycin (100 μg/ml), penicillin(100 units/ml), 2 mM glutamine, and 10% fetal bovine serum (FBS). TheRT7 cell line was cultured using the KGM-2 Bullet Kit (Cambrex). GenomicDNA was extracted therefrom using the Genome DNA Purification kit(Gentra, Minneapolis, Minn.), and RNA was extracted using Isogen (NipponGene), in accordance with manufacturers' instructions.

FIG. 1A shows the strategy for isolating antioncogenic miRNA, the geneexpression level of which has been down-regulated due to methylation oftumor-specific DNA in oral squamous-cell carcinoma, and partial resultsthereof.

At the outset, 18 types of oral squamous-cell carcinoma cell lines andcontrol RT7 cells were used to perform expression profiling of 157 typesof mature miRNA genes. Such expression profiling based on real-timeRT-PCR was carried out using ABI Prism 7500 Fast Real-time PCR (AppliedBiosystems), TaqMan Universal PCR Master Mix (Applied Biosystems), theTaqMan Reverse Transcription Kit (Applied Biosystems), TaqMan Micro RNAAssays (Applied Biosystems), and the Human Panel Early Access Kit(Applied Biosystems) in accordance with the manufacturers' instructions.

Among the 157 types of miRNA genes, miR-124b, miR-144, miR-199-s, andmiR-104 are not currently available for TaqMan Micro RNA Assays and thuswere excluded from the targets of the analysis of the study. In oralsquamous-cell carcinoma, expression levels of miR-154, miR-211, miR-220,miR-302c, and miR-323 genes could not be determined. This is becausesuch gene expression could not be detected via real-time RT-PCR analysisin the RT7 cells for normalization.

In comparison with expression levels of miRNA genes in the RT7 cells,up-regulation in expression of 11 types of miRNA genes (7.4%) (i.e.,expression levels of more than 1.5-fold; more than 66.7% of oralsquamous-cell carcinoma cell lines) and down-regulation of expression of54 types of miRNA genes (36.5%) (i.e., expression levels of less than0.5-fold; more than 66.7% of oral squamous-cell carcinoma cell lines)were observed (Table 1 and Table 2).

TABLE 1 Frequencies of OSCC cell lines with remarkable differences ofmiRNA expression from that in RT7 (≧66.7% of OSCC cell lines) miRNALocus Frequency (%) miRNAs frequently up-regulated in OSCC cell lines(>1.5-fold expression) miR-374 Xq13.2 100.0 miR-340 5q35.3 83.3 miR-224Xq26 83.3 miR-10a 17q21.32 77.8 miR-140 16q22.1 77.8 miR-213 1q31.3 77.8miR-146a 5q33.3 72.2 miR-126 9q34.3 66.7 miR-31 9p21.3 66.7 miR-9miR-9-1, 1q22; miR-9-2, 5q14.3; 66.7 miR-9-3, 15q26.1 miR-9* miR-9-1,1q22; miR-9-3, 15q26.1 66.7 miRNAs frequently down-regulated in OSCCcell lines (<0.5-fold expression) miR-27a 19p13.12 100.0 miR-34b 11q23.1100.0 miR-34c 11q23.1 100.0 miR-203 14q32.33 100.0 miR-302c* 4q25 100.0miR-23a 19p13.12 94.4 miR-27b 9q22.32 94.4 miR-34a 1p36.23 94.4 miR-2151q41 94.4 miR-299 14q32.31 94.4 miR-330 19q13.32 94.4 miR-337 14q32.3194.4 miR-107 10q23.31 88.9 miR-133b 6p12.2 88.9 miR-138 miR-138-1,3p21.33; miR-138-2, 16q13 88.9 miR-139 11q13.4 88.9 miR-223 Xq12 88.9miR-204 9q21.11 88.9 miR-370 14q32.31 88.9 let-7d 9q22.32 83.3 miR-954p16.1 83.3

TABLE 2 miR-302a 4q25 83.3 miR-367 4q25 83.3 let-7g 3p21.1 77.8 miR-23b9q22.32 77.8 miR-128a 2q21.3 77.8 miR-148a 7p15.2 77.8 miR-155 21q21.377.8 miR-200c 12p13.31 77.8 miR-302b 4q25 77.8 miR-368 14q32.31 77.8miR-122a 18q21.31 77.8 miR-371 19q13.41 77.8 let-7a let-7a-1, 9q22.32;let-7a-2, 11q24.1; let-7a-3, 72.2 22q13.31 miR-26b 2q35 72.2 miR-30e-5p1p34.2 72.2 miR-96 7q32.2 72.2 miR-125a 19q13.33 72.2 miR-132 17p13.372.2 miR-200b 1p36.33 72.2 miR-199b 9q34.11 72.2 miR-296 20q13.32 72.2miR-373* 19q13.41 72.2 miR-137 1p21.3 72.2 miR-197 1p13.3 72.2 miR-193a17q11.2 72.2 let-7e 19q13.33 66.7 miR-30d 8q24.22 66.7 miR-331 12q2266.7 miR-342 14q32.2 66.7 miR-338 17q25.3 66.7 miR-199a miR-199a-1,19p13.2; miR-199a-2, 1q24.3 66.7 miR-372 19q13.41 66.7 miR-184 15q25.166.7

Example 2 Analysis of Candidate miRNA and Methylation in OralSquamous-Cell Carcinoma Cell Lines

The human genome database (http://genome.ucsc.edu/) was screened for thepresence of CpG islands in the vicinities of 157 types of miRNA genes.As a result, 21 types of miRNA genes were found to be located on oraround (within 1,000-bp) the CpG islands (Table 3).

TABLE 3 21 miRNAs located on/around CpG islands miRNA Locus miR-9miR-9-1, 1q22; miR-9-3, 15q26.1 miR-9* miR-9-1, 1q22; miR-9-3, 15q26.1miR-34b 11q23.1 miR-92 miR-92-1, 13q31.3; miR-92-2, Xq26.2; miR-92b,1q22 miR-124a miR-124a-1, 8p23.1; miR-124a-2, 8q12.3; miR-124a-3,20q13.33 miR-126 9q34.3 miR-127 14q32.31 miR-129 miR-129-1, 7q32.1;miR-129-2, 11p11.2 miR-132 17p13.3 miR-137 1p21.3 miR-149 2q37.3 miR-15217q21.32 miR-189 9q22.32 (Replaced by miR-24-1) miR-191 3p21.31 miR-193a17q11.2 miR-203 14q32.33 miR-210 11p15.5 miR-219 miR-219-1, 6p21.32;miR-219-2, 9q34.11 miR-320 8p21.3 miR-339 7p22.3 let-7i 12q14.1

Among the 21 types of miRNA genes, the miR-34b, miR-132, miR-137,miR-193a, and miR-203 genes, the expression levels of which aredown-regulated at high frequency in 18 types of oral squamous-cellcarcinoma cells, had attracted attention. In comparison with the RT7cells, the above five miRNA genes (i.e., miR-34b, miR-132, miR-137,miR-193a, and miR-203) exhibited significantly down-regulated expressionlevels in most oral squamous-cell carcinoma cell lines, and thepercentages of such down-regulation were 100% (18/18), 72.2% (13/18),72.2% (13/18), 72.2% (13/18), and 100% (18/18), respectively (FIGS. 1Band C).

In order to inspect whether or not expression of these 5 types of miRNAgenes was suppressed by DNA methylation, oral squamous-cell carcinomacell lines in which these 5 types of miRNA genes had not been expressedwere treated with 1 μM and 10 μM demethylating reagents (5-aza-dCyd) for5 days. RNA was extracted from these cell lines and expression ofcandidate miRNA genes was inspected via real-time RT-PCR (FIG. 1D). As aresult, expression of these genes was found to be recovered viatreatment with 5-aza-dCyd. These results apparently suggest that DNAmethylation is associated with expression suppression of these miRNAgenes.

In order to determine whether or not the state of DNA methylation of the5 types of miRNA genes is correlated with the gene expression patternsin 18 types of oral squamous-cell carcinoma cell lines and in the RT7cell line, the states of DNA methylation of the genes were analyzed byusing the combined bisulfite restriction analysis (COBRA) method. FIG.2A shows the correlation of CpG islands in the 5 types of miRNA genesand the primer positions used for the COBRA method.

Specifically, the EZ DNA methylation kit (Zymo Research, CA, U.S.A.) wasused to treat 2 μg of genomic DNA derived from the oral squamous-cellcarcinoma cell lines in sodium bisulfite at 50° C. overnight, and PCRwas carried out using the primers designed so as to amplify the targetregions (Table 4) (SEQ ID NOs: 1 to 26 of the Sequence Listing).

TABLE 4 Primers for COBRA and bisulfite sequencing miRNA Resion Primerfrom 5′ to 3′ Product size miR-34b 1 Forward GGAGTGGAGGAGTTTTTTGTT 428bp Reverse AAATACCAAACCTCCCCTTC 2 Forward TTAGTTTTAGGGTTTGGGGTT 698 bpReverse TTATAACCACCACAATACAATCAA miR-132 1 ForwardTTTTGGTTTTAGATTGTTTATTG 453 bp Reverse AAACTATTACCTCCAATTCCC 2 ForwardGTTTYGGAAAGTTAATTTTTTG* 599 bp Reverse CCTCACTTTCCTAAAAAAATAAC 3 ForwardGTTATTTTTTTAGGAAAGTGAGG 442 bp Reverse ACTCTACTACTCCRCCTCC** 4 ForwardTTTTGGTTTTAGATTGTTTATTG 408 bp Reverse AAACTATTACCTCCAATTCCC miR-137 1Forward TTTTTTTGTGTTAAGTATTTGATTT 444 bp ReverseAAAAAAATACTACCTTAACAACCA miR-193a 1 Forward AAAGGGAAAATTATTGGGTTT 524 bpReverse AACCCCTCRAACTCCTAA** 2 Forward TTTTAATTTTYGAGGGGTT* 522 bpReverse CAACCCTCCAAAAATTACA 3 Forward GAGGTTTTGGTTTTYGTATTT* 458 bpReverse CCTTCTCCAACRTAAACCT** miR-203 1 Forward TTAGATTTGGGGTAAGTGTTGA405 bp Reverse CCCTCTCACTTCAAAAAAAACT 2 Forward AGTTTTTTTTGAAGTGAGAGGG655 bp Reverse CACCCCCTACCCTACTACAA 3 Forward GTTGTAGTAGGGTAGGGGGT 287bp Reverse ACCCCTAACTATAACTCTAACTCCA *Two kinds of forward primers,which changed a part of “Y” into “C” or “T”, were mixed and used toamplify the bisulfite-modified genomic DNA. **Two kinds of reverseprimers, which changed a part of “R” into “G” or “A”, were mixed andused to amplify the bisulfite-modified genomic DNA.

The resulting PCR products were digested with the BstUI restrictionenzyme (New England BioLabs) or the TaqI restriction enzyme (New EnglandBioLabs). BstUI or TaqI does not digest unmethylated sequences modifiedwith sodium bisulfite, but digests methylated sequences that are notmodified with sodium bisulfite. By utilizing these properties, after PCRfragment was subjected to electrophoresis, the density of the band ofthe methylated fragment and that of the unmethylated fragment wereassayed by densimetry using the MultiGauge2.0 (Fuji Film), and theextent of methylation of the methylated region was represented in termsof percentage. These sequences were subcloned into a TOPO TA cloningvector (Invitrogen), and the nucleotide sequences were determined.

As a result, in the vicinities of the miR-34b, miR-137, miR-193a, andmiR-203 genes, other than the miR-132 gene, hypermethylation of CpGislands or in the vicinity thereof was observed in all the oralsquamous-cell carcinoma cell lines in which down-regulation or quenchingof gene expression has been confirmed (FIG. 2B). In accord with theresults of the COBRA method, hypermethylation in the oral squamous-cellcarcinoma cell lines in which down-regulation or quenching of four typesof miRNA gene expression levels had been confirmed, were verified viabisulfite sequencing (FIG. 2C).

In order to thoroughly analyze miRNA gene expression and DNA methylationin specimens obtained from patients with oral squamous-cell carcinomabased on the above results, 4 types of genes (miR-34b, miR-137,miR-193a, and miR-203) were selected.

Example 3 Analysis of miRNA Expression and Methylation in Specimen fromPatient with Oral Squamous-Cell Carcinoma

Whether or not methylation of four types of miRNA genes has occurred incancerous tissue of a patient with oral squamous-cell carcinoma and thecorrelation between the state of DNA methylation of the four types ofmiRNA genes and the expression patterns in cancerous tissue of a patientwith oral squamous-cell carcinoma were analyzed by using the COBRAmethod and TaqMan real-time RT-PCR analysis.

Regarding cancerous and noncancerous tissue samples of patients withoral squamous-cell carcinoma, 11 cases of frozen samples (T1: 0 cases;T2: 10 cases; T3: 0 cases; T4: 1 case) were obtained with the approvalof the Ethics Committee of Tokyo Dental and Medical University, followedby acquisition of written agreements from patients with oralsquamous-cell carcinoma who had undergone surgery at the Tokyo Dentaland Medical University Hospital, Faculty of Dentistry. The relevantstage was determined in accordance with TNM classification of the UnionInternational Contre le Cancer (UICC).

The COBRA method revealed that hypermethylation of cancer-specific DNAof miR-34b, miR-137, miR-193a, and miR-203 was observed in the canceroustissue of patients with oral squamous-cell carcinoma at frequencies of36.4% (4/11), 63.6% (7/11), 72.7% (8/11), and 45.5% (5/11), respectively(FIG. 3A). In the carcinoma-specific hypermethylation-positive cases ofmiR-137 and miR-193a, apparent fragments derived from alleles that hadbeen methylated in a carcinoma-specific manner were observed.

In many of the carcinoma-specific hypermethylation-positive cases ofmiR-34b and miR-203, the amount of fragments derived from methylatedalleles was very small. This result indicates that the frequency ofcarcinoma-specific hypermethylation in the vicinity of miR-34b ormiR-203 is very low in cancer cells.

When the normal oral cavity mucosa was compared with a cancerous sitevia TaqMan real-time RT-PCR analysis, expression levels of miR-34b,miR-137, miR-193a, and miR-203 were significantly down-regulated (i.e.,27.2% (3/11), 54.5% (6/11), 45.5% (5/11), and 72.7% (8/11) of oralsquamous-cell carcinoma cases) (FIG. 3B). The percentages of oralsquamous-cell carcinoma cases in which both carcinoma-specific DNAhypermethylation and down-regulated or quenched expression of miR-34b,miR-137, miR-193a, and miR-203 were observed were 25% (1/4), 71.4%(5/7), 62.5% (5/8), and 40% (2/5), respectively. This strongly suggeststhat expression of miR-137 and miR-193a is suppressed viacarcinoma-specific hypermethylation in oral squamous-cell carcinoma.Also, bisulfite sequencing of such miRNA in the oral squamous-cellcarcinoma cases apparently verifies the results of the COBRA method(FIG. 3C).

Example 4 Effects of miR-137 and miR-193 a for Suppressing Carcinoma inthe Growth of Oral Squamous-Cell Carcinoma Cell Line

Synthetic double-stranded RNA (dsRNA) comprising miR-137 and miR-193asequences were transiently introduced into the oral squamous-cellcarcinoma cell lines in which down-regulated or quenched expression ofmiR-137 and miR-193 a has been observed. Thus, the effects of miR-137and miR-193a for suppressing cell growth were examined.

Specifically, synthetic double-stranded RNA comprising miR-137 andmiR-193a sequences (i.e., the Pre-miR™ miRNA Precursor Molecule (10 nM,Ambion)) or non-specific miRNA as a control (i.e., Pre-miR™ NegativeControl#1 (Ambion)) was transfected into oral squamous-cell carcinomacell lines using Lipofectamine RNAiMAX (Invitrogen) in accordance withthe manufacturer's instructions. The viable cell counts 24 to 72 hoursafter the transfection were evaluated via WST assay. The results werenormalized with the use of the counts of control cells into whichnonspecific miRNA had been introduced.

The above analysis revealed that introduction of miR-137 and miR-193agenes resulted in significant suppression of growth of oralsquamous-cell carcinoma cells (FIG. 4A). This suggests that miR-137 andmiR-193a have functions of suppressing carcinoma. Also, the TUNELanalysis revealed that lowered capacity for cell growth resulting frommiR-193 a gene introduction was caused by apoptosis (FIG. 4B). The TUNELanalysis was carried out using a TUNEL staining Kit (MEBSTAIN ApoptosisKit Direct; MBL), which can enzymatically label and detect cleavage ofDNA strands in the cells 24 hours after the transfection, in accordancewith the manufacturer's instructions.

1. A method for detecting carcinoma, which comprises detecting malignanttransformation of specimens by employing an up-regulated expressionlevel of at least one gene selected from the group consisting ofmiR-374, miR-340, miR-224, miR-10a, miR-140, miR-213, miR-146a, miR-126,miR-31, miR-9, and miR-9*, and/or a down-regulated expression level ofat least one gene selected from the group consisting of miR-27a,miR-34b, miR-34c miR-203, miR-302c*, miR-23a, miR-27b, miR-34a, miR-215,miR-299, miR-330, miR-337, miR-107, miR-133b, miR-138, miR-139, miR-223,miR-204, miR-370, let-7d, miR-95, miR-302a, miR-367, let-7g, miR-23b,miR-128a, miR-148a, miR-155, miR-200c, miR-302b, miR-368, miR-122a,miR-371, let-7a, miR-26b, miR-30e-5p, miR-96, miR-125a, miR-132,miR-200b, miR-199b, miR-296, miR-373*, miR-137, miR-197, miR-193a,let-7e, miR-30d, miR-331, miR-342, miR-338, miR-199a, miR-372, andmiR-184 as indicators.
 2. The method according to claim 1, whereinchanges in gene expression are detected by using the DNA array method,Northern blotting, RT-PCR, real-time RT-PCR, RT-LAMP, or in situhybridization.
 3. The method according to claim 1, wherein thedown-regulated gene expression level results from methylation of CpGislands or in the vicinity thereof.
 4. The method according to claim 3,wherein the methylation is detected by using the COBRA method, bisulfitesequencing, or Southern blotting.
 5. The method according to claim 1,wherein the down-regulated expression level of at least one geneselected from among miR-34b, miR-132, miR-137, miR-193a, and miR-203 isemployed as an indicator to detect malignant transformation ofspecimens.
 6. The method according to claim 1, wherein the specimen isan oral-cavity-derived cell.
 7. The method according to claim 1, whereinthe carcinoma is oral squamous-cell carcinoma.
 8. An agent forsuppressing carcinoma, which comprises at least one gene selected fromthe group consisting of miR-27a, miR-34b, miR-34c, miR-203, miR-302c,miR-23a, miR-27b, miR-34a, miR-215, miR-299, miR-330, miR-337, miR-107,miR-133b, miR-138, miR-139, miR-223, miR-204, miR-370, let-7d, miR-95,miR-302a, miR-367, let-7g, miR-23b, miR-128a, miR-148a, miR-155,miR-200c, miR-302b, miR-368, miR-122a, miR-371, let-7a, miR-26b,miR-30e-5p, miR-96, miR-125a, miR-132, miR-200b, miR-199b, miR-296,miR-373*, miR-137, miR-197, miR-193a, let-7e, miR-30d, miR-331, miR-342,miR-338, miR-199a, miR-372, and miR-184.
 9. The agent for suppressingcarcinoma according to claim 8, which comprises at least one of miR-34b,miR-132, miR-137, miR-193a, and miR-203 genes.
 10. The agent forsuppressing carcinoma according to claim 8, wherein the gene is bound toor incorporated in a polymer compound.
 11. The agent for suppressingcarcinoma according to claim 10, wherein the polymer compound is aliposome.
 12. The agent for suppressing carcinoma according to claim 8,wherein the carcinoma is oral squamous-cell carcinoma.